Hematopoiesis is organized as a cellular hierarchy initiated by rare self-renewing hematopoietic stem cells (HSC) that also give rise to multipotent progenitors, which in turn produce lineage-committed progenitors and eventually all the terminally differentiated blood cells. It is important to identify the molecular events and pathways regulating hematopoietic stem and progenitor self-renewal, growth, death, and differentiation. However, it has become increasingly clear that studying cell populations only provides average values, and that interrogation of individual cells could reveal the presence of subpopulations and provide insight into these biological processes that are masked at the population level. The main goals for this grant application are to combine the power of microfluidic genomic and epigenomic analysis with the ability to isolate purified hematopoietic stem and progenitor populations to develop a high resolution understanding of hematopoiesis. This proposal aims to use single cell microfluidic PCR to determine the heterogeneity of known populations, and in those cases in which heterogeneity is identified, to functionally characterize the intermediate populations. Next, this proposal aims to use the gene signatures of defined mouse progenitor populations to identify homologues within human hematopoietic tissues. The surface proteins expressed within each type of single cell will be used to separate human cells by FACS, and transplantation into NOG mice will indicate the function of newly identified populations. Deep molecular analyses of these progenitors will be conducted from normal mice and human volunteers to identify candidate gene loci that regulate critical biological functions, including both macro-messenger RNA and microRNA species. The analyses will include identification of new microRNAs, definition of open versus closed chromatin-associated genome sequences, definition of the methylome for each progenitor and comparison with the RNA expression profiles, and when possible analysis of surface expression of the most interesting candidate antigens with monoclonal antibodies and flow cytometry of all populations. Functional assays will be conducted with shRNA to investigate the most interesting genes, and will also include microfluidic nanoculture to assess the effect of soluble factors on progenitor functions. Finally, if it seems possible within the consortium, and if it is within the goals of NHLBI, human bone marrow disorders will be examined from the standpoint of an expanded knowledge of human hematopoietic progenitors.